MmpS5-MmpL5 Transporters Provide Mycobacterium smegmatis Resistance to imidazo[1,2-b][1,2,4,5]tetrazines

The emergence and spread of drug-resistant Mycobacterium tuberculosis strains (including MDR, XDR, and TDR) force scientists worldwide to search for new anti-tuberculosis drugs. We have previously reported a number of imidazo[1,2-b][1,2,4,5]tetrazines–putative inhibitors of mycobacterial eukaryotic-type serine-threonine protein-kinases, active against M. tuberculosis. Whole genomic sequences of spontaneous drug-resistant M. smegmatis mutants revealed four genes possibly involved in imidazo[1,2-b][1,2,4,5]tetrazines resistance; however, the exact mechanism of resistance remain unknown. We used different approaches (construction of targeted mutants, overexpression of the wild-type (w.t.) and mutant genes, and gene-expression studies) to assess the role of the previously identified mutations. We show that mutations in MSMEG_1380 gene lead to overexpression of the mmpS5-mmpL5 operon in M. smegmatis, thus providing resistance to imidazo[1,2-b][1,2,4,5]tetrazines by increased efflux through the MmpS5-MmpL5 system, similarly to the mechanisms of resistance described for M. tuberculosis and M. abscessus. Mycobacterial MmpS5-MmpL5 transporters should be considered as an MDR-efflux system and they should be taken into account at early stages of anti-tuberculosis drug development.